evaluation of permeability and bioavailability of a lead

EVALUATION OF PERMEABILITY AND BIOAVAILABILITY OF A LEAD

ANTILEISHMANIAL COMPOUND BUPARVAQUONE IN ANIMAL

MODELS

by

GANTALA VENKATESH

Thesis submitted in fulfillment of the requirement for the degree of

Doctor of Philosophy

August 2009

DEDICATION

To Lord Venkateshwara Swamy

and

To my late grand parents Rajanna, Govindappa and Govindamma

ii

ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Professor Emeritus

Dr. V. Navaratnam for his guidance, encouragement and patience throughout the

course of this work. His optimism and enthusiasm were always very inspiring.

I am most grateful and enormously indebted to my co-supervisor and Director of the

Centre for Drug Research, Professor Sharif Mahsufi Mansor for his enthusiasm and

continuous support in this project and for giving me the opportunity to carry out this

study in the Centre for Drug Research, Universiti Sains Malaysia. I would also like

to thank my co-supervisor Dr. Surash Ramanathan for his helpful suggestions and

contributions to my work.

My sincere thanks are expressed to Professor N.K. Nair for his invaluable assistance,

support and guidance throughout my research work.

I am grateful to Professor M.I.A. Majid for his expertise guidance and valuable

suggestions in the formulation studies and also for his generous financial support

during my research work.

Special thanks are also expressed to Professor Dr Munavvar Abdul Sattar for his

assistance and for providing laboratory facilities for conducting the permeability

study.

iii

I am most grateful and enormously indebted to my wife and family members for

their love, support and encouragement during this period. They provided a lot of

inspiration and zeal for me to work hard.

I would like to thank my co-researchers, Shubhadra, Lai, Vanessa, Deepa and

Mahabub for spending time in the laboratory discussing ideas in both science and

life. I am grateful to my friends and well wishers especially to Rammohan, Ravi,

Rahamath, Raghu Raj Singh, Mallikarjun, Krishna Murthy, Anand swaroop,

Raghava Naidu, Santosh, Khan, Tito, Srujan, Dinesh, Jignesh Kotecha, Shekar,

Venkatrao, Venku, Raja, Anand, Himani and Phani for their co-operation and moral

support.

Last but not least, my warmest appreciation to all laboratory technicians, especially

Ashokan, Aru, Rahim, Hilman and Zamri for their assistance rendered in the

laboratory. Finally, I would like to thank everybody who was important for the

successful completion of this research work.

Gantala Venkatesh

iv

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv

LIST OF TABLES xii

LIST OF FIGURES xv

LIST OF PLATES xix

LIST OF ABBREVIATIONS & SYMBOLS xx

LIST OF APPENDICES xxii

LIST OF PUBLICATIONS xxiii

ABSTRAK xxiv

ABSTRACT xxvi

CHAPTER 1 : INTRODUCTION

1

1.1 Leishmaniasis 2

1.1.1 Epidemiology 2

1.1.2 Vector transmission 4

1.1.3 Life cycle 4

1.1.4 Clinical manifestations 5

1.1.4 (a) Visceral leishmaniasis or Kala-azar 5

1.1.4 (b) Cutaneous and mucocutaneous leishmaniasis 6

1.1.5 Diagnosis 7

1.1.5 (a) Visceral leishmaniasis 7

1.1.5 (b) Cutaneous leishmaniasis 8

1.1.6 Current chemotherapy for cutaneous and visceral leishmaniasis 8

1.1.6 (a) Pentavalent anitimonials 9

1.1.6 (b) Pentamidine 9

1.1.6 (c) Amphotericin B 10

1.1.6 (d) Other drugs and physical treatment methods 10

1.1.7 Immunotherapy 12

1.1.8 Drugs in development 12

1.1.8 (a) Paramomycin 12

v

1.1.8 (b) Miltefosine 13

1.1.8 (c) Sitamaquine 13

1.1.9 Buparvaquone 14

1.1.10 Buparvaquone-oxime derivatives 16

1.1.11 Water soluble prodrugs of buparvaquone-oxime and

buparvaquone-O-methyl oxime

17

1.1.12 Buparvaquone water soluble phosphate prodrugs

19

1.2 Solubility and gastrointestinal stability

21

1.3 Intestinal permeability 22

1.3.1 Permeability pathways 24

1.3.1 (a) Passive diffusion 25

1.3.1 (b) Carrier-mediated transport or Active transport 25

1.3.1 (c) Carrier-limited transport or Apical efflux 26

1.3.1 (d) Vesicular transport or Pinocytosis 27

1.3.2 In vivo permeability 28

1.3.3 In vitro permeability 29

1.3.3 (a) Caco-2 cell monolayer 29

1.3.3 (b) Intestinal mucosa mounted in ussing chambers 32

1.3.3 (c) Immobilized artificial membrane columns (IAM) and

parallel artificial membrane permeation assay (PAMPA)

33

1.3.4 In situ single-pass perfused rat permeability 36

1.4 Formulation approaches for poorly water soluble drugs 39

1.4.1 Salt formation 40

1.4.2 Reduction of drug particle size 40

1.4.2 (a) Milling techniques 40

1.4.2 (b) High-pressure homogenization 41

1.4.2 (c) Supercritical fluid technology 41

1.4.2 (d) Spray freezing into liquid 43

1.4.2 (e) Controlled precipitation 43

1.4.3 Solid dispersions 44

1.4.4 Inclusion complexes 45

1.4.5 Melt pelletization 46

vi

1.4.6 Lipid based formulations 46

1.4.6 (a) Liposomes 47

1.4.6 (b) Solid lipid nanoparticles 47

1.4.6 (c) Single lipid component solutions 47

1.4.6 (d) Self-emulsifying formulations 48

1.5 Experimental work and scope of the study

51

CHAPTER 2: MATERIALS AND INSTRUMENTS

53

2.1 Materials 53

2.2 Instruments

54

CHAPTER 3: METHODOLOGY

55

3.1 Preliminary stability studies of BPQ in solvents and under UV light 55

3.1.1 Bench stability of BPQ in acetonitrile and methanol at room temperature 55

3.1.2 Stability of BPQ in UV light 55

3.2 Solubility and gastrointestinal fluid stability studies of BPQ 56

3.2.1 Preparation of standard solutions and buffer solutions 56

3.2.1 (a) 0.2 M Hydrochloric acid 56

3.2.1 (b) 0.2 M Potassium biphthalate 56

3.2.1 (c) 0.2 M Monobasic potassium phosphate 56

3.2.1 (d) 0.2 M Potassium chloride 56

3.2.1 (e) 0.2 M Sodium hydroxide 57

3.2.1 (f) Preparation of pH 1.2 buffer solution 57

3.2.1 (g) Preparation of pH 2.0 buffer solution 57

3.2.1 (h) Preparation of pH 3.0 buffer solution 57

3.2.1 (i) Preparation of pH 4.0 buffer solution 57

3.2.1 (j) Preparation of pH 5.0 buffer solution 58

3.2.1 (k) Preparation of pH 6.0 buffer solution 58

3.2.1 (l) Preparation of pH 7.0 buffer solution 58

3.2.1 (m) Preparation of pH 7.5 buffer solution 58

3.2.1 (n) Preparation of simulated gastric fluid 58

3.2.1 (o) Preparation of simulated intestinal fluid 59

vii

3.2.2 Solubility studies 59

3.2.3 Stability studies in simulated gastric and intestinal fluids 59

3.3 Determination of the effect of dimethyl sulfoxide on permeability

markers atenolol and propranolol using rat single-pass intestinal

perfusion method

60

3.3.1 Preparation of buffer solutions 60

3.3.1 (a) Preparation of pH 7.2 perfusion buffer 60

3.3.1 (b) Preparation of pH 7.4 phosphate buffer saline 60

3.3.2 In situ single pass intestinal permeability of atenolol and

propranolol

61

3.3.3 Data analysis and statistics 62

3.3.3 (a) Net water flux

62

3.4 Rat in situ permeability studies for buparvaquone in the presence of

permeability markers

63

3.5 Formulation design and evaluation of self- microemulsifying drug

delivery system (SMEDDS) for buparvaquone

64


3.5.2 Pseudo-ternary phase diagram study 64

3.5.3 Preparation of selected SMEDDS formulations and determination

of maximum drug loading in the formulations

65

3.5.4 Droplet size analysis 65

3.5.5 Stability of the prepared SMEDDS in SGF and SIF 67

3.5.6 Emulsification time 67

3.5.7 In vitro dissolution test 68

3.6 Evaluation of bioavailability and pharmacokinetic parameters of

buparvaquone

68

3.6.1 Preparation of intravenous formulation 68

3.6.2 Study protocol 68

3.6.3 Analysis of BPQ in plasma samples 69

3.6.4 Pharmacokinetic analysis 71

viii

CHAPTER 4 : DEVELOPMENT AND VALIDATION OF RP-HPLC-UV

METHODS FOR BUPARVAQUONE

73

4.1 Development and validation of RP-HPLC-UV method for

determination of buparvaquone in solubility and gastrointestinal

fluid stability studies

73

4.1.1 Preparation of stock and working standard solutions 73

4.1.2 Preparation of calibration and quality control samples 73

4.1.3 Liquid chromatographic conditions 73

4.1.4 Absorption spectrum of buparvaquone 74

4.1.5 Method Validation 74

4.1.5 (a) Specificity 74

4.1.5 (b) Linearity 74

4.1.5 (c) Accuracy and precision 74

4.1.5 (d) Stability studies 75

4.1.6 Results and Discussion 75

4.1.6 (a) Absorption spectrum of buparvaquone 75

4.1.6 (b) Specificity 75

4.1.6 (c) Linearity 79

4.1.6 (d) Accuracy and precision 80

4.1.6 (e) Stability studies

80


determination of permeability markers atenolol and propranolol in

rat in situ perfusion method

82

4.2.1 Preparation of 0.02 M ammonium acetate buffer 82




4.2.5 Method Validation 84




ix


4.2.5 (e) Recovery 85








determination of buparvaquone, atenolol, propranolol, quinidine

and verapamil in rat in situ perfusion method

92




4.3.4 Method validation 94




4.3.4 (d) Recovery 95

4.3.4 (e) Stability studies 96


4.3.5 (a) Method development 96

4.3.5 (b) Specificity 101




4.3.5 (f) Stability studies

110

4.4 Development and validation of RP-HPLC-UV method with solid

phase extraction for determination of buparvaquone in human and

rabbit plasma

112




x

4.4.4 Sample preparation 113

4.4.5 Method validation 114


4.4.5 (b) Carryover check 114




4.4.5 (f) Stability studies 116

4.4.5 (g) Dilution integrity 117

4.4.6 Partial validation in rabbit plasma 117


4.4.7 (a) Method development 118

4.4.7 (b) Choice of internal standard 120

4.4.7 (c) Sample preparation 121

4.4.7 (d) Specificity 122

4.4.7 (e) Carryover check 122

4.4.7 (f) Linearity 125

4.4.7 (g) Accuracy and precision 127

4.4.7 (h) Recovery 127

4.4.7 (i) Stability studies 127

4.4.7 (j) Dilution integrity 130

CHAPTER 5 : RESULTS

132

5.1 Preliminary stability studies of BPQ in solvents and under UV light 132

5.1.1 Bench stability of BPQ in acetonitrile and methanol at room

temperature

132

5.1.2 Stability of BPQ in UV light 135

5.2 Solubility and gastrointestinal fluid stability studies of buparvaquone 137


5.2.2 Stability study in gastrointestinal fluids 137

xi

5.3 Determination of the effect of dimethyl sulfoxide on permeability

markers atenolol and propranolol using rat single - pass intestinal

perfusion method

138

5.4 Rat in situ permeability studies for buparvaquone in the presence of

permeability markers

141

5.5 Formulation design and evaluation of self-microemulsifying drug

delivery system (SMEDDS) for buparvaquone

143


5.5.2 Pseudo-ternary phase diagram 144

5.5.3 Drug loading 150

5.5.4 Droplet size analysis 152

5.5.5 Emulsification time 155

5.5.6 In vitro dissolution test 157

5.6 Evaluation of bioavailability and pharmacokinetic parameters of

buparvaquone

158

5.6.1 Pharmacokinetic analysis 158

5.6.2 Bioavailability of BPQ

161

CHAPTER 6 : DISCUSSION AND CONCLUSIONS

162

6.1 Discussion 162

6.2 Conclusions

175

CHAPTER 7 : SUGGESTIONS FOR FUTURE WORK

177

BIBLIOGRAPHY

179

APPENDICES

199

PUBLICATIONS

xii

LIST OF TABLES

Page

1.1 Leishmaniasis disease patterns and common organisms in different geographical locations.

3

3.1 Composition of SMEDDS formulations selected from the phase diagram with 2:1 ratio of Cremophor EL to Labrasol.

66

3.2 Composition of SMEDDS formulations selected from the phase diagram with 4:1 ratio of Cremophor EL to Labrasol.

66

4.1 Linearity parameters for buparvaquone. Mean ± S.D, n=3.

79

4.2 Experimental values of mean concentration, %R.E. and %R.S.D for the validation parameters of buparvaquone.

81

4.3 Preparation of working standard solutions for atenolol and propranolol.

83

4.4 Linearity parameters for atenolol and propranolol. Mean ± S.D, n=3.

88

4.5 Experimental values of mean concentration, %R.E. and %R.S.D for the validation parameters of atenolol and propranolol.

90

4.6 Recovery studies of atenolol and propranolol in blank rat perfusion buffer. Mean ± S.D, n=5.

91

4.7 Preparation of working standard solutions for quinidine, verapamil and buparvaquone.

93

4.8 Linearity parameters for BPQ and permeability markers. Mean ± S.D, n=3.

105

4.9 Intra and inter-day precision and accuracy of atenolol, propranolol, quinidine, verapamil and BPQ.

107

4.10 Recovery studies of atenolol, propranolol and quinidine in blank rat perfusion buffer. Mean ± S.D, n=5.

108

4.11 Recovery studies of verapamil and BPQ in blank rat perfusion buffer. Mean ± S.D, n=5.

109

4.12 Stability studies for BPQ and permeability markers. Mean ± S.D, n=5.

111

xiii

4.13 Preparation of BPQ secondary working standard solution in acetonitrile.

113

4.14 Linearity parameters of BPQ in human plasma. Mean ± S.D, n=6.

125

4.15 Mean accuracy of the calibration standards (n=6) for BPQ in human plasma.

126

4.16 Intra and inter-day precision and accuracy for BPQ in human and rabbit plasma.

128

4.17 Recovery studies of BPQ in human and rabbit plasma. Mean ± S.D, n=3.

129

4.18 Stability studies of BPQ in human plasma. Mean ± S.D, n=3.

131

5.1 Degradation of BPQ in acetonitrile and methanol at room temperature.

132

5.2 Photolytic degradation of BPQ in acetonitrile and methanol at room temperature.

135

5.3 Solubility of BPQ in the pH range of 1.2 to7.5 at 37°C in buffer solutions. Mean ± S.D, n=3.

137

5.4 BPQ stability in simulated gastric and intestinal fluids. Mean ± S.D, n=3.

138

5.5 Peff values for permeability markers atenolol and propranolol at various concentration of dimethyl sulfoxide (1-5%)

140

5.6 Rat in situ intestinal Peff values for atenolol, propranolol and BPQ. Mean ± S.D, n=6.

142

5.7 Solubility of BPQ at 37°C in various vehicles. Mean ± S.D, n=3.

143

5.8 Solubility of BPQ in SMEDDS containing a mixture of Cremophor EL to Labrasol ratios, 2:1 (F1 to F8) and 4:1 (F9 to F16). Mean ± S.D, n=3.

150

5.9 Composition of SMEDDS formulations with maximum drug loading of BPQ in the 2:1 ratio of Cremophor EL to Labrasol.

151

5.10 Composition of SMEDDS formulations with maximum drug loading of BPQ in the 4:1 ratio of Cremophor EL to Labrasol.

151

xiv

5.11 Droplet size of formulations with Cremophor EL to Labrasol ratio, 2:1. Mean ± S.D, n=3.

152

5.12 Droplet size of formulations with Cremophor EL to Labrasol ratio, 4:1. Mean ± S.D, n=3.

153

5.13 Effect of drug loading on the droplet size for formulations F2, F3, F10 and F11 in SGF and SIF.

155

5.14 Emulsification time for formulations of 2:1 and 4:1 Cremophor EL to Labrasol ratio in SGF (pH 1.2) at 37 °C.

156

5.15 Pharmacokinetic parameters of BPQ with intravenous administration dose of 1.0 mg/kg of BPQ. Mean ± S.D, n=3.

158

5.16 Pharmacokinetic parameters of BPQ with oral administration dose of 3.0 mg/kg of BPQ. Mean ± S.D, n=3.

160

xv

LIST OF FIGURES

Page

1.1 Global distribution of leishmaniasis. 3

1.2 Leishmania life cycle.

5

1.3 Chemical structures of (a) meglumine antimoniate, (b) sodium stibogluconate, (c) pentamidine and (d) amphotericin B.

11

1.4 Chemical structures of (a) miltifosine and (b) sitamaquine.

14

1.5 Chemical structure of buparvaquone (BPQ).

15

1.6 Chemical structures of buparvaquone oxime derivatives.

17

1.7 Chemical structure of water soluble prodrugs of BPQ (a) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-hydroxy-[1, 4] naphthoquinone-1-(O-phosphono oxime) and (b) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-hydroxy-[1, 4] naphthoquinone-1-(O-phosphon ooxymethyloxime).

18

1.8 Chemical structure of water soluble prodrugs of BPQ (a) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-phosphono-[1,4]naphothaquinone-1-(O-methyloxime) and (b) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-phosphonooxymethoxy - [1,4]naphthoquinone-1-(O-methyloxime).

18

1.9 Schematic flow chart for formation of BPQ from buparvaquone-oxime phosphate prodrugs.

19

1.10 Chemical structures of water soluble phosphate prodrugs of BPQ: (a) buparvaquone-3-phosphate and (b) 3-phosphonooxymethyl-buparvaquone.

20

1.11 Biopharmaceutical Classification System.

22

1.12 Anatomical details of intestinal mucosa.

24

1.13 Different routes of drug entry from the intestine into the blood stream.

26

1.14 Effect of various concentrations of dimethyl sulfoxide on permeability markers in the PAMPA model.

36

1.15 Rat in situ intestinal perfusion setup.

37

4.1 Absorption spectrum (200-400 nm) of BPQ (50 µg/ml) in acetonitrile.

76

4.2 Representative chromatogram of blank simulated gastric fluid (SGF) sample.

77

xvi

4.3 Representative chromatogram of blank simulated intestinal fluid (SIF) sample.

77

4.4 Representative chromatogram of SGF sample at 4.5 µg/ml of BPQ (90 min).

78

4.5 Representative chromatogram of SIF sample at 4.5 µg/ml of BPQ (180 min).

78

4.6 Typical linearity curve of BPQ in the range of 100-2000 ng/ml.

79

4.7 Representative chromatograms of A) blank perfusion sample B) standards spiked in blank perfusion sample, the peaks of interest are atenolol (Aten) and propranolol (Ppn) and (C) rat perfusate sample collected between 15-20 min.

87

4.8 Typical linearity curves for atenolol (Aten) and propranolol (Ppn) in the range of 15-120 and 5-60 µg/ml.

88

4.9 Chromatograms at three different pH of 0.02 M ammonium acetate and acetonitrile in the ratio of 30:70 (v/v): A) pH 3.0, B) pH 3.5 and C) pH 4.0. The peaks of interest are atenolol (Aten), quinidine (Quin), propranolol ( Ppn), verapamil (Ver) and BPQ.

97

4.10 Chromatograms at three different mobile phase ratios of 0.02 M ammonium acetate (pH 3.5) and acetonitrile: A) 25:75 (v/v) B) 30:70 (v/v) and C) 35:65 (v/v). The peaks of interest are atenolol (Aten), quinidine (Quin), propranolol (Ppn), verapamil (Ver) and BPQ.

99

4.11 Representative chromatograms using optimized chromatographic conditions of A) blank perfusion sample, B) standards spiked in blank perfusion sample, the peaks of interest are atenolol (Aten), quinidine (Quin), propranolol (Ppn), verapamil (Ver) and BPQ and C) methanol as organic solvent in the mobile phase instead of acetonitrile.

100

4.12 Typical linearity curve for atenolol in the range of 15-120 µg/ml.

102

4.13 Typical linearity curve for propranolol in the range of 5-60 µg/ml.

103

4.14 Typical linearity curve for quinidine in the range of 0.8-8.0 µg/ml.

103

4.15 Typical linearity curve for verapamil in the range of 10-100 µg/ml.

104

4.16 Typical linearity curve for BPQ in the range of 200-1200 ng/ml.

104

4.17 Chemical structure of lovastatin (LOVA).

120

xvii

4.18 Representative chromatograms: (A) blank human plasma and (B) blank rabbit plasma.

123

4.19 Representative chromatograms: (A) BPQ and lovastatin spiked in blank rabbit plasma (LLOQ-50 ng/ml) (B) 15 min sample following an intravenous administration of 1.0 mg/kg BPQ to rabbit.

124

4.20 Linearity curve for BPQ concentrations between 50 to 800 ng/ml.

125

5.1 Degradation profile of BPQ in acetonitrile at room temperature.

133

5.2 Degradation profile of BPQ in methanol at room temperature.

133

5.3 Representative chromatogram of the degradation of BPQ in methanol at room temperature after 13 days.

134

5.4

Representative chromatogram of the degradation of BPQ in acetonitrile at room temperature after 13 days.

134

5.5

Representative chromatogram of the degradation of BPQ in acetonitrile under UV light after 19 hours.

136

5.6 Representative chromatogram of the degradation of BPQ in methanol under UV light after 19 hours.

136

5.7 Effect of various concentration of DMSO on the rat in situ intestinal permeability of atenolol (▫) and propranolol (▪).

140

5.8 Pseudo-ternary phase diagram consisting of water, Capryol 90 and mixture of Cremophor EL and Labrasol (surfactant: cosurfactant ratio of 1:1 w/w).

145


146


147


148

5.12 An overlay pseudo-ternary phase diagram consisting of water, Capryol 90, Cremophor EL and Labrasol at ratio of surfactant: cosurfactant 1:1, 2:1, 3:1 and 4:1 w/w.

149

xviii

5.13 Effect of Cremophor EL on droplet size for formulations containing Cremophor EL to Labrasol ratio, 2:1.

153

5.14 Effect of Cremophor EL on droplet size for formulations containing Cremophor EL to Labrasol ratio, 4:1.

154

5.15 Emulsification time in SGF (pH 1.2) at 37 °C versus %w/w of Cremophor EL in formulations F1 to F16.

156

5.16 In vitro dissolution profile of BPQ in SGF (pH 1.2). Mean ± SD, n=6.

157

5.17 In vitro dissolution profile of BPQ in SIF (pH 6.8). Mean ± SD, n=6.

157

5.18 Plasma concentration versus time profile in rabbits following an intravenous dose of 1.0 mg/kg of BPQ. Mean ± SD, n=3.

159

5.19 Plasma concentration versus time profile in rabbits following an oral dose of 3.0 mg/kg of BPQ. Mean ± SD, n=3.

160

xix

LIST OF PLATES

3.1 Holding rabbit in upright position 69

3.2 Prior to dosing keeping the mouth holder 70

3.3 SMEDDS formulation was administered by a syringe with oral gavage tube through the mouth holder

70

xx

LIST OF ABBREVIATIONS & SYMBOLS

ANOVA = Analysis of variance

AUC = Area under curve

BALB/c = Albino Laboratory Breed Strain of Mice

BCS = Biopharmaceutical classification system

BSA = Bovine serum albumin

BPQ = Buparvaquone

CL = Cutaneous leishmaniasis

Caco-2 = Cell line: human colon carcinoma cells

CV = Coefficient of variation

DMSO = Dimethyl sulfoxide

FDA = Food and Drug Administration

HPLC = High performance liquid chromatography

IAM = Immobilized Artificial Membrane

IV = Intravenous

LOQ = Limit of quantification

MDR = Multidrug resistance

MRP = Multidrug resistance associated protein

MDCK = Madin-Darby canine kidney cells

mg = Milligram

MW = Molecular weight

MCL = Mucocutaneous leishmaniasis

NTDS = Neglected tropical diseases

NWF = Net water flux

PAMPA = Parallel artificial membrane permeation assay

P-gp = P-glycoprotein

PBS = Phosphate buffer saline

PEG = Polyethylene glycol

RD = Relative difference

RE = Relative error

xxi

RPM = Rotation per minute

RSD = Relative standard deviation

SPIP = Single pass intestinal perfusion technique

SD = Standard deviation

SEDDS = Self-emulsifying drug delivery systems

SMEDDS = Self-microemulsifying drug delivery systems

SIF = Simulated intestinal fluid

SGF = Simulated gastric fluid

SPSS = Statistical procedures for social science

SPE = Solid phase extraction

TDR = Training in tropical diseases

TEER = Transepithelial electrical resistance

UV = Ultra violet

v/v = Volume in volume

VL = Visceral leishmaniasis

WHO = World Health Organization

w/w = Weight in weight

xxiv

KAJIAN KETELAPAN DAN BIOKEPEROLEHAN BUPARVAQUONE,

SUATU SEBATIAN PELOPOR ANTILEISMANIAL DALAM MODEL

HAIWAN

ABSTRAK

Leishmaniasis merupakan suatu kumpulan penyakit parasit yang disebabkan oleh

genus Leishmania yang kini adalah endemik di lapan puluh lapan negara. Penyakit

ini wujud dalam dua bentuk utama, iaitu leishmaniasis kulitan dan visera. Sebatian

antimoni pentavalensi merupakan kaedah yang paling umum digunakan untuk

mengubati semua penyakit jenis ini. Kajian in vitro dan in vivo menunjukkan

buparvaquone (BPQ) sebagai suatu sebatian yang berpotensi untuk digunakan

terhadap jangkitan Leishmania donovi. Dalam membangunkan formulasi oral BPQ,

penentuan ciri-ciri biofarmaseutikal seperti keterlarutan dan ketelapan drug perlu

dikaji terlebih dahulu. Kajian kestabilan di peringkat awal menunjukkan bahawa

sampel BPQ yang dilarutkan dalam metanol dan asetonitril membentuk sebatian

degradasi yang belum dikenalpasti apabila didedahkan kepada cahaya. Kadar

degradasi juga didapati meningkat apabila didedahkan kepada cahaya ultra

lembayung. Kajian keterlarutan BPQ dalam pelbagai jenis larutan penimbal piawai

(pH 1.2 hingga 7.5) telah dikaji dan didapati keterlarutan tertinggi tidak melebihi 210

ng/ml. BPQ didapati stabil dalam bendalir gastrik dan usus simulasi, di mana

degradasi adalah kurang dari 5%. Keputusan ini mencadangkan bahawa BPQ akan

tetap stabil dalam saluran perut usus sebelum penyerapan berlaku. Ketelapan atenolol

and propranolol telah dikaji dalam model tikus in situ pada kepekatan DMSO yang

berbeza (1-5%). Tiada perubahan signifikan (p>0.05) ke atas nilai purata petunjuk-

petunjuk ketelapan tersebut. Nilai yang didapati ini adalah sejajar dengan nilai

ketelapan yang dilaporkan sebelum ini. Keputusan kajian ini menunjukkan integriti

xxv

membran tisu usus tikus tidak dipengaruhi oleh kehadiran DMSO sehingga sebanyak

5%. Drug yang berketerlarutan rendah dalam air sering memberi masalah dalam

kajian ketelapan kerana keterlarutan yang rendah dan penyerapan tidak spesifik

menjadi punca kepada anggaran ketelapan yang tidak tepat. Oleh kerana BPQ adalah

suatu sebatian yang berketerlarutan rendah dalam air, maka kajian ketelapan

dilakukan dengan kehadiran 5% DMSO sebagai pelarut bersama. Nilai ketelapan

purata bagi BPQ adalah 0.912 x 10-4 cm/s dan ini menunjukkan bahawa ia adalah

sebatian jenis kelas II, di mana ia adalah berketelapan tinggi walaupun

berketerlarutan rendah berpandukan kepada Sistem Klasifikasi Biofarmaseutikal

(SKB). Sistem Penyampaian Drug Swa-Mikroemulsifikasi (SPDSM) merupakan

suatu formulasi oral yang boleh digunakan untuk drug kelas II SKB untuk

meningkatkan keterlarutan dan biokeperolehan. Perkembangan dan penyaringan

formulasi oral SPDSM telah dilakukan berdasarkan kepada keputusan kajian

keterlarutan pelbagai eksipien, gambarajah fasa untuk penentuan kawasan swa-

mikroemulsifikasi, analisis saiz butiran dan masa emulsifikasi. Pelarutan in vitro dan

kajian farmakokinetik telah dilakukan ke atas formulasi yang optimum yang terdiri

daripada Capryol 90 (9.82 %w/w) dan campuran Cremophor EL/Labrasol (4:1)

(88.40 %w/w) dan BPQ 1.78 %w/w. Formulasi ini menghasilkan saiz butiran tyang

terkecil berukuran 18 nm. Kaedah analisis fasa terbalik HPLC-UV yang berbeza

telah diperkembangkan dan disahsahihkan untuk penentuan BPQ dalam pelbagai

matriks biologi. Kajian biokeperolehan BPQ telah dilakukan dalam arnab jantan

putih New Zealand. Parameter farmakokinetik telah ditentukan untuk kedua-dua

intravena dan formulasi oral bagi BPQ dan nilai-nilai min Cmax, Tmax dan AUC(0-36)

adalah 999.83 ng/ml, 0.25 jam, 2872.71 ng.jam/ml dan 171.74 ng/ml, 7.33 jam,

3456.38 ng.jam/ml masing-masing. Biokeperolehan mutlak adalah 40.10 %.

xxvi

EVALUATION OF PERMEABILITY AND BIOAVAILABILITY OF A LEAD

ANTILEISHMANIAL COMPOUND BUPARVAQUONE IN ANIMAL

MODELS

ABSTRACT

Leishmaniasis is a group of parasitic disease caused by the protozoan parasites of the

genus Leishmania and is currently endemic in 88 countries. This disease occurs in

two major forms, cutaneous and visceral leishmaniasis. Pentavalent antimonials are

the most widely used therapy for all forms of the disease. In in vitro and in vivo

studies buparvaquone (BPQ) has been found to be a promising lead compound for

the treatment of Leishmania donovani infections. The successful development of oral

drug delivery formulation for BPQ requires determination of the biopharmaceutical

properties mainly solubility and permeability. In preliminary stability studies, the test

samples of BPQ in methanol and acetonitrile form unknown degradation compounds

when exposed to the light and the rate of degradation was increased in the presence

of UV light. BPQ solubility studies were carried out in various standard buffer

solutions (pH 1.2 to 7.5) and the solubility was found to be not more than 210 ng/ml.

BPQ was stable in both simulated gastric and intestinal fluids with < 5% degradation.

These results suggest that BPQ would be stable in the gastrointestinal tract prior to

absorption. Permeability of atenolol and propranolol were carried out in rat in situ

model at various concentrations of dimethyl sulfoxide (DMSO, 1-5%). There was no

significant alteration (p>0.05) in the mean values of the permeability markers. The

values agreed with previously reported permeability values. The results indicate that

the membrane integrity of the rat intestinal tissues was not affected by the presence

of up to 5% DMSO. Poorly water soluble drugs often cause difficulties in

permeability studies due to their poor solubility limitation and non-specific

xxvii

adsorption which lead to inaccurate estimation of permeability. Since BPQ is a

poorly water soluble compound the permeability study was carried out in the

presence of 5% DMSO as a co-solvent. The mean permeability value for BPQ was

0.912 x10-4 cm/s indicating that it is a class II compound which is a highly permeable

and poorly water soluble compound according to the biopharmaceutical classification

system (BCS). Self-Microemulsifying drug delivery system (SMEDDS) is a feasible

oral formulation approach for BCS class II drugs since it increases their solubility

and bioavailability. Oral SMEDDS formulation development and screening was done

based on results obtained from solubility in various excipients, phase diagrams for

identifying microemulsification region, droplet size analysis and emulsification time.

In vitro dissolution and pharmacokinetic studies were performed with the optimized

formulation which is composed of Capryol 90 (9.82% w/w) and mixture of

Cremophor EL/Labrasol (4:1) (88.40% w/w) and BPQ (1.78% w/w). This

formulation yielded the smallest droplet size of 18 nm. Different RP-HPLC-UV

analytical methods were developed and validated for determining BPQ in the various

biological matrices. Bioavailability study of BPQ was conducted in healthy white

New Zealand male rabbits. Pharmacokinetic parameters were calculated for both i.v

and the optimized oral formulation of BPQ and the mean Cmax, Tmax and AUC (0–36)

were 999.83 ng/ml, 0.25 hour, 2872.71 ng.hour/ml and 171.74 ng/ml, 7.33 hour,

3456.38 ng.hour/ml respectively. Absolute bioavailability was found to be 40.10%.

CHAPTER 1

INTRODUCTION

Tropical diseases are infectious diseases that occur uniquely in tropical and

subtropical regions. In the Special Programme for Research and Training in Tropical

Diseases of the World Health Organization (WHO/TDR), various

international/national bodies and philanothropic foundations focus on neglected

infectious diseases that mainly affect poor and marginalized populations in the

developing regions of America, Asia, and Africa. Neglected Tropical Diseases

(NTDS) cause approximately 534,000 deaths annually. These include bacterial,

protozoan and helminth tropical infectious diseases such as leishmaniasis, African

trypanosomiasis, dengue fever, malaria, schistosomiasis, tuberculosis, chagas

disease, leprosy, lymphatic filariasis and onchocerciasis. Although leprosy,

tuberculosis, cholera and yellow fever do not occur in the tropics their highest

incidence in the tropics justify their inclusion in tropical diseases (Nwaka and

Hudson, 2006; Hotez et al., 2007).

Some tropical diseases are very rare but they may occur in sudden epidemics, such as

the ebola hemorrhagic fever, marburg haemorrhagic fever and lassa fever. There are

hundreds of different tropical diseases which are less known such as oropouche

fever, lobomycosis, west nile disease, labrea fever, rocio disease, mapucho

hemorrhagic fever, trachoma, guinea worm disease and chikungunya (Tropical

disease, 2008).

1

1.1 Leishmaniasis

1.1.1 Epidemiology

Leishmaniasis is a major widespread parasitic disease and is caused by the protozoan

haemoflagellates of the genus Leishmania. There are at least 20 species of leishmania

which are transmitted by 30 species of sandflies and each may cause disease specific

to the species and the host response. It is endemic in many parts of the world and the

geographical distribution of the disease is shown in Figure 1.1. There are several

forms of the disease named by their clinical presentation including cutaneous

leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis

(MCL). Each of these forms of disease is caused by different species of sandflies

found in different regions of the world. Table 1.1 shows the common species that

cause leishmaniasis in different geographical locations. Old World CL in humans is

associated with members of L. aethiopica, L. major, and L. tropica complex while L.

mexicana and L. braziliensis complex transmit New World CL. VL is caused by L.

donovani and L. infantum in the Old World regions while L. chagasi is primarily

responsible for visceral disease in the New World (Herwaldt, 1999; Bailey and

Lockwood, 2007; Davies et al., 2003).

The disease is prevalent worldwide and threatens 350 million men, women and

children, affecting 88 countries of which 72 are developing countries and 13 of them

are among the least developed. High occurrence of VL is found in 65 countries and

90% of VL cases occur in poor rural and suburban areas of 5 countries namely

Bangladesh, India, Nepal, Sudan and Brazil. Majority of CL cases occur in 7

countries namely Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria

2

Figure 1.1 Global distribution of leishmaniasis (Davies et al., 2003).

Table 1.1 Leishmaniasis disease patterns and common organisms in different

geographical locations.

Clinical disease Leishmaniasis species Geographical location

Cutaneous leishmaniasis L. tropica complex: L. tropica, L. aethiopica and L. major. Old World

L. mexicana complex: L. Mexicana, L.pifanoi , L. amazonensis, L. garnhami and L. venezuelensis .

New World

L. braziliensis complex: L. peruviana , L.guyanensis L. panamensis L. lainsoni and L. colombiensis.

New World

L. infantum Old World L. chagasi New World

Visceral leishmaniasis L. donovani complex: L. donovani, L. infantum Old World

L. chagasi New World L. tropica Old World L. amazonensis New World

Mucocutaneous leishmaniasis

L. braziliensis complex: L. braziliensis , L.guyanensis and L. panamensis New World

L. Mexicana New World L. tropica and L. major Old World

Old World: N.Africa, South Asia, Middle East, Tunisia Ethiopia, Kenya, India,

Sudan, Bangladesh, China, Central and West Asia, Central and South America, Mexico, Belize, Guatemala and Ecuador.

New World: Venezuela, Brazil, Peru, French Guiana, Guyana, Surinam, Panama and

Costa Rica

3

and 90% of MCL occur in Bolivia, Brazil and Peru (Bailey and Lockwood, 2007;

Piscopa and Mallia, 2006).

1.1.2 Vector transmission

The leishmania species belongs to the class Kinetoplastida and family

Trypanosomatida. The sandflies (2-3 mm long) which are the vectors that transmit

the disease are from genus Phlebotomus in the Old World and Lutzomyia and

Psychodopygus in the New World. Sandflies are relatively weak, noiseless fliers;

they rest in dark and moist places and are typically more active in the evening and

night. These vectors are nocturnal by nature and it is the females that prey on blood.

The parasites are introduced into the host (human) through the bite of an infected

female sandfly (Herwaldt, 1999).

1.1.3 Life cycle

During the life cycle, the parasite exists in two forms; amastigote form in human and

promastigote form in sand fly. The various stages of the leishmania life cycle is

shown in Figure 1.2. The female sandflies inject the infective stage metacyclic

promastigotes (elongated motile and with an anterior flagellum) during blood meals

(1). Metacyclic promastigotes that reach the puncture wound are phagocytized by

macrophages (2) and undergoes transformation into amastigotes (3). Amastigotes (2-

3 µm in diameter) multiply by binary fission in the macrophages and proceed to

infect other mononuclear phagocytic cells (4). These amastigotes will affect different

tissues of the body, depending on which leishmaniasis species is involved. These

differing tissue specificities cause the different clinical manifestations of the various

4

Figure 1.2 Leishmania life cycle (Source: http://en.wikipedia.org.).

forms of leishmaniasis. Sandflies become infected during blood meals from an

infected host when they ingest macrophages infected with amastigotes (5, 6). In the

sandfly midgut, the parasites differentiate into promastigotes (7), which multiply,

differentiate into metacyclic promastigotes and migrate to the pharynx and proboscis

(8) (Leishmaniasis, 2007; Chappuis et al., 2007).

1.1.4 Clinical manifestations

1.1.4 (a) Visceral leishmaniasis or Kala-azar

VL comprises a broad range of manifestations of infection. The clinical symptoms

are characterized by fever, weakness, night sweats and weight loss and these

symptoms continue over weeks to months. Lymphadenopathy, hyperpigmentation

5

and hepatosplenomegaly (spleen much larger than the liver) are of common

occurrence in VL. Anaemia, leucopenia, or thrombocytopenia, and

hypergammaglobulinaemia are also frequently manifested symptoms. Untreated or

progressing disease can produce profound cachexia, multisystem disease, bleeding

from thrombocytopenia, susceptibility to secondary infections and death may rapidly

follow (Chappuis et al., 2007; Singh et al., 2006).

1.1.4 (b) Cutaneous and mucocutaneous leishmaniasis

Cutaneous forms of the disease normally produce skin ulcers on the exposed parts of

the body such as face, arms and legs. It starts as a papule at the site of a bite,

increasing in size, causing ulcer. It may take 3-18 months to heal in typical cases.

The incubation period lasts from 2 weeks to months and cases up to 3 years have

been reported in Old World CL. In New World CL, the incubation period is usually

2-8 weeks (Piscopa and Mallia, 2006). In the Old World CL most of the lesions

appear in the form of nodules, papules or nodule ulcers, but most New World CL

may begin with ulcerative lesions. In MCL the lesions affect the mucocutaneous

membranes of the nose, mouth, and throat cavities causing partial or total destruction

of the surrounding tissues (Bailey and Lockwood, 2007; Desjux, 2004). The

incubation period for MCL is 1-3 months, but it may occur many years after the

initial cutaneous ulcer has healed. Mucosal type occurs in South American cases of

CL (espundia). This causes difficulty in eating and an increased risk for secondary

infection, which carries considerable mortality (Piscopa and Mallia, 2006).

6

1.1.5 Diagnosis

1.1.5 (a) Visceral leishmaniasis

Diagnosis of VL is routinely based on microscopic detection of amastigotes in

smears of tissue aspirates or biopsy samples. An aspirate or biopsy of the bone

marrow is frequently the tissue of choice, with sensitivities in the 55-97% range. The

gold standard for diagnosing VL is parasite identification in tissue smears with

splenic aspirate being more sensitive than bone marrow or lymph node aspirates.

Lymph node aspirate smears (sensitivity 60%) or biopsy, and splenic aspirates

(sensitivity 97%) may also be taken for diagnosis. The parasite may also be cultured

from microscopy negative tissue samples on special media such as Novy, McNeal or

Nicolle medium or inoculated into animals such as hamsters (Singh et al., 2006).

Direct Agglutination Test (DAT) is commonly used diagnosis for VL and it is easy

to use in the field. DAT has been shown to be sensitive and quite specific (sensitivity

of 91-100% and a specificity of 72-100%). However its use is limited due to the non

availability of standardized antigen and adaptability especially in the rural centres.

Immunochromatographic rapid strip testing of blood from a finger prick for

leishmanial rK39 antibody has been used successfully in the field of serodiagnosis

with a sensitivity of 90-100% in patients showing symptoms. Leishmania DNA can

be detected in the tissue aspirates and peripheral blood using polymerase chain

reaction (PCR), with sensitivities in the range of 70-93% in peripheral blood. PCR

and in situ hybridisation have been used to amplify DNA sequences of the parasite in

biopsy material and is highly sensitive for the diagnosis of VL and can be used to

monitor therapeutic response (Piscopo and Mallia, 2006; Chappuis et al., 2007;

Singh et al., 2006). A new commercial latex agglutination test (KATEX) was used

for preliminary testing to detect the leishmanial antigen in patient’s urine with

7

sensitivity in the range of 68-100% and 100% specificity. However, this test is still

under the preliminary phase evaluation and its commercialization is still far from

reality (Singh et al., 2006; Davies et al., 2003).

1.1.5 (b) Cutaneous leishmaniasis

Tissue aspirate, scraping and fresh skin biopsies are employed frequently to obtain

samples. Microscopical examinations of skin scrapings or biopsy specimens taken

from the edge of lesions are used for the diagnosis of CL. This method is rapid and

economical but has limited sensitivity, especially for chronic lesions. Monoclonal

antibodies are used for identification, but a direct analysis of clinical specimens is

better achieved using PCR. Antibody detection is less sensitive due to the lack of

significant antibody production in CL. Other means of diagnosing CL include the

Montenegro (leishmanin) skin test, which detects specific cutaneous delayed-type

hypersensitivity. It involves intradermal injection of L.mexicana antigen, and

monitoring for a local reaction. Limitations of this test include its inability to

distinguish between current and past infection, as well as report of false positivity in

other skin infections (Piscopo and Mallia, 2006; Bailey and Lockwood, 2007).

1.1.6 Current chemotherapy for cutaneous and visceral leishmaniasis

Currently available drugs which are used for treatment of leishmaniasis include

meglumine antimonite (Glucantime, Aventis), sodium stibogluconate (Pentostam,

Glaxo SmithKline), amphotericin B(Bristol-Myers Squibb), amphotericin B lipid

formulation (AmBiosome®, Gilead) and pentamidine (Aventis).

8

1.1.6 (a) Pentavalent antimonials

For the past 50 years, treatment for both CL and VL was mainly dependent on

pentavalent anitimonials, sodium stibogluconate (Pentostam) and meglumine

antimoniate (Figure 1.3). The antimonials were introduced in 1945 and they are

effective for the treatment of some forms of leishmaniasis. In recent years it has been

found that there is an increase in the parasite resistance for these drugs. These

antimonials are less effective against VL and MCL in the Bihar state of India

because of poor pharmacokinetics and variation in the species sensitivity, all these

factors limiting the drugs usefulness. The standard therapy for both VL and CL

involves daily intravenous injections for 20 days of sodium stibogluconate or

meglumine antimoniate at a dose of 5 mg on first day and increased to maximum

total dose of 850 mg/day (Fernando et al., 2001; Croft and Coombs, 2003; Ouellette

et al., 2004).

1.1.6 (b) Pentamidine

Pentamidine isethionate (Figure 1.3) is an antiprotozoal agent introduced in 1952 and

is used as a second-line antileishmanial drug for both CL and VL when antimonials

are resistant to the parasite. The effective treatment for CL was found to be at the

dose of 2 mg/kg administered intravenously on alternate days for 14 days. The

treatment for VL which produces a cure rate of 70% requires a dose of 4 mg/kg on

alternate days for a total of 40 days. However pentamidine has limitations in its use

as an antileishmanial drug due to considerable toxicity such as hypotension,

hypoglycemia, and nephrotoxicity. (Fernando et al., 2001; Ouellette et al., 2004).

9

1.1.6 (c) Amphotericin B

Amphotericin B (Figure 1.3) is an antifungal agent recommended as a second-line

drug for the treatment of VL, CL and MCL. Amphotericin B was administered at a

dose of 0.75-1.0 mg/kg on alternate days for 30 days. Amphotericin B usage is

limited by its narrow therapeutic index and the side effects are fever, hypotension,

liver failure and blood dyscrasias. (Singh et al., 2006). However the efficacy of

Amphotericin B has been improved by the development of a liposomal formulation

which is rapidly effective and non toxic and has been approved by FDA for treatment

of VL in adults. It is given by IV at 3 mg/kg/day for 30 days for children with drug

resistance VL (Croft et al., 2005; Kafetzis et al., 2005; Murray, 2004).

1.1.6 (d) Other drugs and physical treatment methods

12% methlybenzethonium chloride in soft paraffin is used for CL and the duration of

application depends on the strain of parasite. Oral medications such as dapsone,

allopurinol, ketaconazole, levamisole and rifampicin are also used for the treatment

of CL. The dose of dapsone, allopurinol, ketaconazole, levamisole and rifampicin is

1-2 mg/kg/day for six weeks, 20 mg/kg/day for 15 days, 10 mg/kg in three divided

doses for 4 weeks, 150 mg/day on two successive days each week for 8 weeks and

10 mg/kg for 6 -12 weeks respectively. However these drugs have side effects such

as hepatocellular changes, allergic reactions, vomiting, nausea and abdominal pain.

In case of rifampicin, the body fluids become red. Allopurinol and ketaconazole or

both in combination are also used for the treatment of VL (Fernando et al., 2001).

10

(a)

(b)

(c)

(d)

Figure 1.3 Chemical structures of (a) meglumine antimoniate, (b) sodium

stibogluconate, (c) pentamidine and (d) amphotericin B.

11

Physical treatments like cryotherapy and thermotherapy have been used for the

treatment of cutaneous lesions for duration of 4 weeks with a success rate from 77%

to 100%. However their use is limited because of availability of equipment,

secondary infections, slower healing of large scars and poor compliance (Bailey and

Lockwood, 2007).

1.1.7 Immunotherapy

Active VL causes a defect in the immune system by causing a deficiency in

interferon-γ production in the body. In this case, rational therapeutic approach of

treatment would involve the stimulation of the immune system. For AIDS patients

with VL, pentavalent antimoniate and interferon-γ with a dose of 100-400 µg/m2

body surface/day is recommended for a course of 10-40 days. However the treatment

is still in its early stages and requires further development. (Fernando et al., 2001).

1.1.8 Drugs in development

1.1.8 (a) Paramomycin

Paramomycin is a non toxic aminoglycoside aminocyclitol antibiotic which is an

effective for the treatment of both VL and CL and is currently in clinical trials. A

suitable monotherapy regimen of paramomycin sulphate is 16 mg/kg, administered

intramuscularly for 21 days (Singh et al., 2006). An ointment with 15%

paramomycin and 0.5% gentamicin has been shown to be 100% effective for CL in

BABL/c mice (Davis and Kedzierski, 2005).

12

1.1.8 (b) Miltefosine

Miltefosine (Figure 1.4) is an alkylphosphocholine originally developed as an

anticancer agent. Miltefosine has shown efficacy in several in vitro and in vivo

experimental models as an effective oral drug for the treatment of VL. These

findings led to clinical trials and registration of miltefosine for oral treatment of VL

in India in March 2002 and for CL in Colombia in 2005. In phase 3 trials miltefosine

cured 94 % of VL patients with a dose of 2.5 mg/kg for 28 days. Cutaneous lesions

were healed with topical application of 6% miltefosine ointment for 2-5 weeks (Croft

and Engel, 2006; Croft and Coombs, 2003). A study was conducted in VL male

patients in India to evaluate the patients’ fertility and results showed that there were

no clinically relevant changes on patients’ fertility. The major limitation of

miltefosine is teratogenicity and is therefore not recommended for women in child-

bearing age. (Sindermann and Engel, 2006).

1.1.8 (c) Sitamaquine

Sitamaquine (Figure1.4) is a primaquine analogue (8-aminoquinolene) and was

developed as an alternative oral drug by Walter Reed Army Institute of

Research/Glaxo SmithKline. An initial phase I/II study showed 67% cure rate in

patients treated with a dose of 2 mg/kg/day for a duration of 28 days in Brazil and

92% cure rate when treated with 1.7 mg/kg daily for 28 days in Kenya for VL (Davis

and Kedzierski , 2005; Dietze et al., 2001). Sitamaquine has mild toxicity and causes

methemoglobinaemia (Croft et al., 2006).

13

(a)

(b)

Figure 1.4 Chemical structures of (a) miltefosine and (b) sitamaquine.

1.1.9 Buparvaquone Buparvaquone (BPQ), 4-hydroxy-3- [(4-tert-butylcyclohexyl) methyl] naphthalene-1,

2-dione (Figure 1.5) is a second-generation hydroxynaphthoquinone related to

parvaquone. It has novel features that make it a promising compound for the therapy

and prophylaxis of all forms of theileriosis. In in vivo studies BPQ showed

approximately twenty times more activity than parvaquone and it has been tested

widely against Theileria annulata, T. parva and T. sergenti both in laboratory studies

and in field trials. In an acute oral toxicological study in rat, the observed LD50 value

for BPQ was more than 8000 mg/kg which showed BPQ has low toxicity (McHardy,

1988). Currently BPQ has been formulated as a solution for intramuscular injection

(Butalex, Coopers Animal Health). The Butalex injection is a safe, convenient and an

additional alternative drug product to the existing antitheilerial products (McHardy et

al., 1985).

14

Figure 1.5 Chemical structure of buparvaquone (BPQ).

As part of a programme to find new drugs for treatment of leishmaniasis, a series of

hydroxynaphthoquinones were tested against Leishmania donovani in vitro and in

vivo. In vitro experiments showed BPQ to be 100 fold more active against L.

donovani amastigotes than other hydroxynaphthoquinones with ED50 value of 0.005

µM. However, in vivo experiments performed in BABL/c mice model with oral and

subcutaneous routes with a dose of 100 mg/kg BPQ for five days resulted in

suppression of only 35 to 62% liver amastigotes. This low activity was considered to

be due to its poor distribution from the site of injection and poor oral bioavailability

due to insufficient absorption (Croft et al., 1992). Butalex was injected

intramuscularly to seven dogs which were symptomatic and parasitologically

positive for canine visceral leishmaniasis with 4 doses of 5 mg/kg over a 12 day

period. Only two dogs showed minor clinical improvement and as a consequence it

was concluded that BPQ was ineffective for the treatment of symptomatic visceral

leishmaniasis (Vexenat et al., 1998).

BPQ has very poor aqueous solubility (<0.03 µg/ml), high lipophilicity (at pH 3.0

Log D -7.02) and a pKa value of 5.70. The low solubility and high lipophilicity are

the most probable reasons for the low in vivo activity of BPQ against leishmaniasis

in oral drug delivery. Furthermore BPQ metabolizes more actively with mouse and

15

dog hepatic microsomes in the presence of NADPH when compared to human

hepatic microsomes. There is no reported pathway available in literature on the

metabolism of BPQ (Mantyla et al., 2004a; Croft et al., 1992).

A pharmacokinetic study has been reported for BPQ and parvaquone in cattle in

which BPQ was administered into the neck muscles at a dose of 2.5 mg/kg. There

was no significant variation in absorption and distribution rates for both drugs but a

wide variation was observed in the mean plasma concentrations for BPQ (50 to 280

ng/ml). The average elimination half life was 26.44 hours for BPQ and there was no

intravenous data for calculating bioavailability (Kinabo and Bogan, 1988).

1.1.10 Buparvaquone-oxime derivatives

Mantyla et al. (2004a) reported that BPQ was active in in vitro studies against both

amastigotes and promastigotes in low nanomolar range but in vivo results were

disappointing. This finding led to the synthesis of various oxime derivatives of BPQ

as novel antileishmania compounds. The buparvaquone oxime containing C=NOH

group undergoes a CYP-450 dependent oxidative cleavage to BPQ (Figure 1.6).

Buparvaquone oxime has better aqueous solubility (5.42 µg/ml) when compared to

BPQ and other oxime derivatives such as buparvaquone-O-methlyloxime and O-

methly-buparvaquone oxime. Buparvaquone oxime derivatives have shown low in

vitro activity against intracellular leishmania donovani amastigotes when compared

to BPQ. The lower activity of these buparvaquone oxime compounds is most

probably due to their slow conversion to BPQ during the experiment.

16

Figure 1.6 Chemical structures of buparvaquone oxime derivatives.

1.1.11 Water soluble prodrugs of buparvaquone-oxime and buparvaquone-O-

methlyoxime

The phosphate prodrug approach is a widely used strategy to improve aqueous

solubility of drugs containing hydroxyl group. Thus researchers combined the oxime

and phosphate prodrug strategies for improving the aqueous solubility of BPQ.

Novel water soluble phosphate prodrugs of buparvaquone-oxime and buparvaquone-

O-methlyoxime were synthesized and all the prodrugs have good aqueous solubility

(Figures 1.7 and 1.8). The most promising prodrug is 3-(trans-4-tert-butyl-cyclo

hexylmethyl)-2-hydroxy-[1,4]naphthoquinone-1-(O-phosphonooxymethlyoxime) or

(Phosphonooxymethyl-buparvaquone-oxime) (Figure 1.7 b). It has good aqueous

solubility (4.2 mg/ml) and chemical stability over the pH range of 3.0-7.4 and rapidly

releases the parent buparvaquone oxime via chemical or enzymatic bioconversion by

alkaline phosphatases (t1/2 4.1 min) (Figure 1.9). Parent buparvaquone-oxime has

been suggested to be able to efficiently permeate through the intestinal wall and form

BPQ after oxidative metabolism (Mantyla et al., 2004b).

17

(a) (b)

Figure 1.7 Chemical structure of water soluble prodrugs of BPQ (a) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-hydroxy-[1, 4] naphthoquinone-1-(O-phosphono oxime) and (b) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-hydroxy-[1,4] naphthoquinone-1-(O-phosphon ooxymethyloxime).

(a) (b)

Figure 1.8 Chemical structure of water soluble prodrugs of BPQ (a) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2-phosphono-[1,4]naphothaquinone-1-(O-met hyloxime) and (b) 3-(trans-4-tert-butyl-cyclohexylmethyl)-2- phosphonoox ymethoxy - [1,4]naphthoquinone-1-(O-methyloxime).

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Phosphonooxymethyl-buparvaquone-oxime

Enzymatic hydrolysis

Alkaline phosphatases

Buparvaquone-oxime

P450 oxidation

Buparvaquone

Figure 1.9 Schematic flow chart for formation of BPQ from buparvaquone-oxime phosphate prodrugs.

1.1.12 Buparvaquone water soluble phosphate prodrugs

Prodrugs release the parent drug during or after absorption by enzymatic or chemical

hydrolysis. However a phosphate group that is directly attached to a hydroxyl group

is not always cleaved enzymatically due to steric hindrance. Therefore researchers

focussed on designing enzymatically labile phosphate prodrugs which release the

drug as BPQ by hydrolysis. Aqueous solubility of the prodrugs buparvaquone-3-

phosphate and 3-phosphonooxymethyl-buparvaquone is more than 3.5 mg/ml over

the pH range of 3.0-7.4 (Figure 1.10). These phosphate prodrugs are sufficiently

stable in the gastrointestinal tract before their absorption and rapidly hydrolyze

(t1/2=1.2 and 3.8 min) to the parent compound in the presence of alkaline phosphatase

(Mantyla et al., 2004).

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(a) (b)

Figure 1.10 Chemical structures of water soluble phosphate prodrugs of BPQ: (a) buparvaquone-3-phosphate and (b) 3-phosphonooxymethyl-buparvaquone

In in vitro activity studies phosphate prodrugs have been shown to have potent

activity in the nanomolar range against the various species of the promastigotes and

amastigotes due to their conversion to BPQ. Of these two phosphate prodrugs 3-

phosphonooxymethyl-buparvaquone is the more potent antileishmanial compound.

Human skin permeation of BPQ can also be significantly improved by using

phosphate prodrugs. 3-phosphonooxymethyl-buparvaquone was shown to be rapidly

converted to the parent drug during skin permeation studies (Mantyla et al., 2004;

Garnier et al., 2007).

BPQ and phosphate prodrugs were evaluated by in vivo studies for both VL and CL.

BPQ (hydrous gel and water-in-oil emulsion formulations) and phosphate prodrugs

(anhydrous gel formulation) significantly reduce the parasite burden and lesion size

when compared to the untreated control for CL. These findings suggest that BPQ and

phosphate prodrugs are effective for the treatment of CL. In visceral model BPQ was

prepared as a solution in isopropyl myristate and polyethylene glycol 400 and the

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phosphate prodrugs were prepared in 50 mM sodium acetate buffer (pH 5.0).

Prodrugs caused a significant decrease in the liver parasite burden when compared to

BPQ. These results suggest that BPQ and phosphate prodrugs need to be investigated

further for the treatment of VL (Garnier et al., 2007a).

1.2 Solubility and gastrointestinal stability

In the drug discovery process, the lead candidates must have proper physicochemical

properties in order to have a better chance of success in the development. Solubility,

pKa (ionization constant) and lipophilicity are the most fundamental

physicochemical properties of a drug candidate and the determination of these

parameters are essential in both in silico and in vitro models. Based on

Biopharmaceutical Classification System (BCS), the drug bioavailability mainly

depends on the aqueous solubility and the ability of molecule to permeate through

the biological membrane (Figure 1.11). Many pharmacologically active compounds

fail to become pharmaceutical drugs because of poor bioavailability and

unacceptable physicochemical properties. As a consequence, solubility and

permeability have been incorporated into drug discovery programs (Delaney, 2005;

Huuskonen, 2001).

BCS recommends determining the solubility of drug substances under physiological

pH conditions. The experiments should be carried out at 37 ± 1 º C in aqueous media

with a pH range of 1.2 to 7.5. To obtain accurate solubility data, the pH solubility

profile of the compound should be determined based on its pKa. Solubility should be

determined at pH= pKa, pH= pKa +1, pH= pKa -1, pH 1 and 7.5 (USFDA, 2000).

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Class I

High Solubility

High Permeability

Class II

Low Solubility

High Permeability

Class III

High Solubility

Low Permeability

Class IV

Low Solubility

Low Permeability

Figure 1.11 Biopharmaceutics Classification System.

Prior to permeability studies, it is necessary to determine the stability of the drug in

gastric and intestinal (G.I) fluids. These results will assist in obtaining accurate

permeability values. When conducting permeability studies in in vivo or in situ

models there is possibility for degradation of the drug in the G.I fluids prior to

absorption which will give rise to erroneous permeability values. Hence it is

necessary to determine the stability of the drug in both gastric (pH 1.2) and intestinal

(pH 6.8) conditions.

USFDA, (2000) recommends conducting G.I stability by incubating the drug

solution in these fluids at 37º C for a period that is representative of in vivo drug

contact with these fluids. The experiments should be conducted for 1 hour in gastric

fluids and for 3 hours in intestinal fluids.

1.3 Intestinal permeability

A major objective in the pharmaceutical industry is to develop new drugs with good

oral bioavailability. The new chemical entities are first selected on the basis of their

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pharmacological activity followed by sequential profiling to assess the solubility,

absorption, distribution, metabolism, elimination and toxicity for lead selection and

optimization (Trapani et al., 2004; Li, 2004).

Drug bioavailability mainly depends on the aqueous solubility and the ability of the

molecule to permeate through the biological membrane. Hence, the extent of in vivo

absorption of the drug can be assessed based on solubility and in vitro permeability.

Thus determination of intestinal permeability is one essential requirement for

assessing the oral bioavailability of a new drug candidate at the early drug discovery

stage (Volpe, 2008).

Absorption is defined as the movement of unchanged drug from the site of

administration into the systemic circulation. The small intestine, which consists of

duodenum, jejunum and ileum is the most important site for drug absorption in the

gastrointestinal tract. The small intestine permits the absorption of organic and

inorganic chemicals/nutrients such as sugars, amino acids, lipids and vitamins and

but prevents the entry of xenobiotics and digestive enzymes (Venkateshwarlu, 2004).

Intestinal membrane is not a unicellular structure but it is a group of unicellular

membranes parallel to one another. For drugs and nutrients to be absorbed into the

systemic circulation they must penetrate the intestinal mucosa. The intestinal mucosa

consists of three layers: muscularis mucosa, lamina propia and epithelial cell layer. A

basement membrane is present below the epithelial cell layer and lamina propia lies

between the basement membrane and muscularis mucosa, which contains connective

tissue, blood and lymph vessels (Figure 1.12). The epithelial cell layer which

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possesses kerkrings, villi and microvilli provides a large surface area for drug

absorption (Venkateshwarlu, 2004; Hiremath, 2000).

Figure 1.12 Anatomical details of intestinal mucosa (Source: Venkateshwarlu, 2004).

Drug molecules penetrate most of the barriers in the intestine and reach the lamina

propia. From this site, molecules either may diffuse through the blood stream or

penetrate the central lacteal and enter the lymph. Most of the drugs reach the

systemic circulation via the blood stream of the capillary network in the villi. The

villi are highly and rapidly perfused by the blood stream with the blood flow rate of

approximately 500 to 1000 times greater than lymph flow rate. Therefore the

lymphatic system will account for only a small fraction of total amount of drug

absorbed compared to the systemic circulation. The capillary and lymphatic vessels

are permeable to most low molecular weight and lipid-soluble compounds. However

capillary membrane is a more substantial barrier than the central lacteal for the

permeation of large molecules (Venkateshwarlu, 2004; Hiremath, 2000).

1.3.1 Permeability pathways

For orally administered drugs to exert pharmacological activity, they must dissolve

and permeate through several cellular membranes in the intestinal wall in order to

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evaluation of permeability and bioavailability of a lead

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